CN105675554A - Fluoro-spectrophotometry method for detecting total flavones in health-caring foods - Google Patents

Abstract

The invention discloses a fluoro-spectrophotometry method for detecting flavones in health-caring foods, which includes the following steps: adding an ethanol water solution to a sample to extract the total flavones to obtain an extracted sample liquid; and adding an acetic acid-sodium acetate buffer liquid to the sample liquid with uniform mixing, and adding a 95% aluminium nitrate ethanol solution for complexation, adding a fluorescent sensitizer triethanolamine water solution, and measuring the fluorescent value at excitation wavelength of 410 nm and emission wavelength of 515 nm, and calculating the content of the total flavones in the sample. The method has good selectivity and high sensitivity and is accurate and reliable.

Description

A kind of fluorimetry detecting total flavones in protective foods

Technical field

The invention belongs to analysis detection field, it relates to the detection of functional component Flavonoid substances in protective foods, especially relate to the total flavones in fluorimetry detection protective foods.

Background technology

Flavonoid compound, referred to as flavones, is the general name of a class natural compounds with benzene a pair of horses going side by side pyranose ring structure. Many herbal medicine and food exist, has multiple biological activity, there is antibacterial, anti-inflammatory, reducing blood-fat, antianginal, anti-oxidant and eliminate free radical, improve the effects such as body immunity. Therefore, flavonoid compound is often applied in protective foods. At present, more taking total flavonoids substance as the healthcare products of main function on market.

The content analysis of total flavones adopts ultraviolet spectrophotometry and high performance liquid chromatography usually. Wherein spectrophotometry is the easiest. (Wang Guangya is edited, China Light Industry Press, 2002 to test discovery " protective foods functional component detection method " through applicant. 29th page) described in method steps more loaded down with trivial details, expend time in longer, it relates to decompress filter, degreasing, cross the process such as post. When sample contains a large amount of unknown complex composition, color developing effect is undesirable, affects absorbancy, the accuracy that final impact measures. " protective foods inspection and assessment technical specifications " (Ministry of Health of the People's Republic of China, version in 2003. 308th page) described in method regulation with methanol solution extract, cross polyamide column, and standard substance direct methanol dissolve, preparation reference liquid, but post measure, cause the rate of recovery on the low side; Use the organic solvent such as methyl alcohol, benzene in a large number simultaneously, not environmentally, harmful HUMAN HEALTH. Also there is the relevant report adopting high performance liquid chromatography detection total flavones, but analytical instrument cost is expensive.

Fluorescence derivatization be can with in some compound structures some functional group produce intense fluorescence reagent. Reaction is to these reagent requirements:

(1) should be able to when comparing mitigation with measured matter fast quantification react;

(2) fluorescent derivative generated should have satisfactory stability;

(3) before launching or during original position derivatize product with excessive derivatization reagent is necessary can be very easily separated;

(4) derivatization reagent itself should not have fluorescence.

The present invention, according to the principle of Flavonoid substances and aluminium salt formation fluorescent complex, adds trolamine, can significantly strengthen fluorescent characteristic, moreover it is possible to strengthen its fluorescent stability.

The present invention, by repetition test, establishes a kind of fluorimetry detecting total flavones in protective foods. The method selectivity is good, highly sensitive, accurately and reliably.It is suitable for government's quality testing department, manufacturer, third party feeler mechanism to the detection of total flavones in protective foods.

Summary of the invention

It is an object of the invention to provide a kind of method measuring determination of total flavonoids in protective foods, the method invented can overcome now methodical deficiency, it is possible to significantly improves now methodical sensitivity, the present invention by the following technical solutions:

(1) sample adds extraction solution water-bath refluxing extraction total flavones, filters, obtain extracting solution;

(2) add damping fluid 0.5-1.5mL, regulate the pH value of solution to 3.0-5.0;

(3) add aluminum salt solution 0.2-0.5mL, form binary aluminum ion-flavonoid binary complex system;

(4) fluorescence sensitivity agent 1-3mL is added, mixed even, fixed appearance;

(5) at excitation wavelength lambda ex410nm, emission wavelength lambda em515nm measures fluorescent value, calculates the content of total flavones in protective foods.

In step (1), extraction solution used is aqueous ethanolic solution, and volume fraction is 50%-70%.

In step (2), damping fluid used is 5.88mmol/L acetic acid-sodium acetate buffer solution, and its pH is 3.0-5.0, and volume used is 0.5-1.5mL;

Aluminium salt used in step (3) is aluminum nitrate or aluminum chloride, it is preferable that aluminum chloride, and massfraction is 5-10%, and solvent is 95% ethanolic soln;

In step (4), fluorescence sensitivity agent is trolamine, mass concentration 5-10% used, and volume is 1-3mL; (6) instrumental analysis parameter

Using molecular fluorescence photometer, excitation wavelength lambda ex410nm, emission wavelength lambda em515nm, slit width is 5nm.

The technique effect of the present invention is:

1. pre-treating process is simply effective, it is not necessary to carry out decompress filter, degreasing, cross post, etc., save time, environmental protection, avoid traditional method and use the hazardous and noxious substances such as methyl alcohol, benzene, Sodium Nitrite;

2. the method is quick. Organic solvent extraction, filters, it is not necessary to crosses column purification, adds aluminium salt, damping fluid, fluorescence sensitivity agent, the upper machine of fixed appearance;

3. the method is accurately feasible, highly sensitive. The detection of traditional method is limited to 3.5 μ g/mL; The detection limit of present method total flavones reaches 0.05 μ g/mL, in concentration range at 0.05 μ g/mL-30 μ g/mL, has good linear relationship.

In a word, the method is easy, quick, environmental protection, reliable, detection limit is low, avoid and use the dangerous harmful reagent such as methyl alcohol, benzene and nitrite, the defect that complex steps in tradition spectrophotometry sample, the length that expends time in, color interference, color developing effect are undesirable can be overcome. The fluorescence intensity of binary complex system can be significantly improved, be the 6-7 of conventional surfactant when doing fluorescence sensitivity agent doubly.

Accompanying drawing illustrates:

Fig. 1: flavones fluorescence contrast figure (5.0 μ g/mL, λ ex=410nm).

A: add trolamine fluorogram; B: former fluorogram

Fig. 2: several tensio-active agent and trolamine flavones fluorescence contrast figure (5.0 μ g/mL, λ ex=410).

A marks liquid+10% trolamine 2mL; B marks liquid+polysorbas20;

C. liquid+0.2% Sodium dodecylbenzene sulfonate is marked; D. liquid+0.2% CETRIMIDE POWDER is marked

Embodiment

Here is that the present invention is described in further details.

Embodiment 1:

The comparison that fluorescence intensity is affected by 2 kinds of tensio-active agent fluorescence sensitivity dosage and trolamine fluorescence sensitivity agent

Analytical procedure:

Precision takes rutin standard substance (examining and determine institute purchased from the biological product of China's medicine) 5mg, is mixed with the standardized solution that concentration is 50 μ g/mL, accurately draws standardized solution 0,0.1mL respectively, 0.2mL, 0.5mL, 1.0mL, 2.0mL, is placed in 10mL volumetric flask, adds 10%AlCl₃Solution 0.5mL, is settled to 5mL with the ethanolic soln of 60%, adds acetic acid-sodium acetate buffer solution 1mL, mixed even, add each tensio-active agent 1mL, 60% ethanolic soln is settled to 10mL, mixed even, obtaining series concentration is 0 μ g/mL, 0.5 μ g/mL, 1.0 μ g/mL, 2.5 μ g/mL, 5 μ g/mL, 10 μ g/mL, upper machine measures.

Using Japan's Shimadzu RF5301 molecular fluorescence photometer, at excitation wavelength lambda ex410nm, emission wavelength lambda em515nm, slit width is 5nm.

Its comparing result is in table 1:

Table 1: the comparison that fluorescence intensity is affected by several tensio-active agent and trolamine

Remarks: 1 mark liquid+trolamine 0.2mL; 2 mark liquid+0.2% Sodium dodecylbenzene sulfonatees;

3 mark liquid+0.2% CETRIMIDE POWDER;

The flavones fluorescence contrast figure (5.0 μ g/mL, λ ex=410) of 2 kinds of conventional surfactant fluorescence sensitivity agent and trolamine fluorescence sensitivity agent is shown in Fig. 2.

Embodiment 2:

The comparison of typical curve and preparation

Analytical procedure:

1. add fluorescence sensitivity agent: precision takes rutin standard substance (examining and determine institute purchased from the biological product of China's medicine) 5.0mg, is mixed with the standardized solution that concentration is 50 μ g/mL, accurately draws standardized solution 0,0.1,0.2,0.5,1.0,2.0, it is placed in 10mL volumetric flask, adds 10%AlCl₃Solution 0.5mL, is settled to 5mL with the ethanolic soln of 60%, adds acetic acid-sodium acetate buffer solution 1mL, mixed even, add 10% triethanolamine solution 2mL, 60% ethanolic soln is settled to 10mL, mixed even, obtaining series concentration is 0 μ g/mL, 0.5 μ g/mL, 1.0 μ g/mL, 2.5 μ g/mL, 5 μ g/mL, 10 μ g/mL, upper machine measures. Condition determination: excitation wavelength lambda ex=410nm, emission wavelength lambda em=515nm, slit width is 5nm.

2. do not add fluorescence sensitivity agent. Not adding triethanolamine solution, all the other operating process are the same, increase by 25 μ g/mL standard points. Its comparing result is in table 2:

Table 2 fluorescence sensitivity sense agent is on the impact of fluorescent value

Remarks: 1 for adding the fluorescent value of fluorescence sensitivity sense agent trolamine;

2 for not adding the fluorescent value of any sensitizer

Embodiment 3

Method measures contrast

Analytical procedure

Test 1 is according to " protective foods functional component detection method " chapter 2 Section three: in the measuring method of total flavones, spectrophotometry measures; Test 2 employing sample ethanolic soln water-bath refluxing extraction 2h, cold filtration, gets filtrate 1mL and is undertaken by concrete embodiment 1 standard curve making processes. Test have chosen certain capsule, certain oral liquid and certain electuary, and its Comparative result is as follows:

Result in table three three kinds of different sorts healthcare products

Embodiment 4

Recovery of standard addition is tested

Analytical procedure

In order to the accuracy of validating experiment, take to add sample absorption method and carried out recovery test. Precision takes the trial-product 0.5g of known content, and this sample is 0.173g/100g through fluorometric determination content. Take standard substance 0.1g and prepare rutin standard solution, add 0.50mg/mL rutin standardized solution 1.0mL, 2.0mL, 3.0mL, extracting solution is settled to 25mL and gets 1.0mL, measures fluorescence intensity according to optimum experimental condition, and measure fluorescence intensity according to optimum test condition, by the general flavone content in regression equation calculation sample, calculate the rate of recovery, in table four.

The result of table four recovery of standard addition

Learn by table four, add be designated as 1-3mL time, average recovery rate is 94.5%.

Above-described embodiment is used for illustrative purposes only, and is not the restriction to patent of the present invention; It should be appreciated that for the those of ordinary skill of this area, when not departing from present inventive concept scope, it is also possible to make various change and modification, these all belong to protection scope of the present invention; Therefore, all impartial changes done with the claims in the present invention scope, with modifying, all should belong to the coverage of the claims in the present invention.

Claims (4)

1. detecting a fluorimetry for total flavones in protective foods, its feature comprises the steps:

(1) adding extraction solution water-bath refluxing extraction total flavones in sample, direct filtration, obtains extracting solution;

(2) add 5.88mmol/L acetic acid-sodium acetate buffer solution 0.5-1.5mL, regulate the pH value of solution to 3.0-5.0;

(3) add aluminum nitrate or aluminum chloride 95% ethanolic soln 0.2-0.5mL that massfraction is 5%-10%, form binary aluminum ion-flavonoid binary complex system;

(4) fluorescence sensitivity agent 1-3mL is added, mixed even, fixed appearance;

(5) at excitation wavelength 410nm, emission wavelength 515nm place measures fluorescent value, calculates the content of total flavones in protective foods.

2. method according to claims 1, it is characterised in that, extraction solution used is 50%-70% aqueous ethanolic solution.

3. method according to claims 1, it is characterised in that, damping fluid used is acetic acid-sodium acetate.

4. method according to claims 1, it is characterised in that, fluorescence sensitivity agent used is trolamine, and mass concentration is 5%-10%.